JPH0627109A - Immunological measurement method and reagent kit therefor - Google Patents

Abstract

PURPOSE:To enable highly sensitive measurement by having an immobilized substance, a first nucleic acid combination and a marker second nucleic acid reacted with a substance to be measured to measure a marker volume in produced immunological complex. CONSTITUTION:An immobilized substance is a combination of a combining substance and a water insoluble carrier, wherein a silicic acid inorganic carrier or the like having a functional group such as an amino group on a surface is preferable as the water insoluble carrier. First and second nucleic acids are both one-chain nucleic acid selected from deoxyribonucleic acid(DNA) or ribonucleic acid(RNA) whose base sequences are complementary to each other. A substance having the same immunological reaction characteristics as those of a substance to be measured is combined with the first nucleic acid and a marker is combined with the second nucleic acid, respectively. The second nucleic acid may be one-chain DNA or RNA corresponding to only a part of the first nucleic acid, and a plurality of the second nucleic acids can be combined with a molecule of the first nucleic acid using shorter nucleic acid than the first nucleic acid. Therefore, since immunological complex is produced in a form that numerous marker second nucleic acids are combined on the first nucleic acid combination, measurement sensitivity is improved.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay method and an immunoassay reagent. More specifically, it relates to a heterogeneous immunoassay and a reagent kit for immunoassay.

[0002]

2. Description of the Related Art Conventionally, in a heterogeneous sandwich immunoassay, a substance to be measured (hereinafter referred to as "measurement substance") is bound to a water-insoluble carrier by an antibody or an antigen against the "measurement substance", And a method of reacting a labeled antibody or antigen against the “measurement substance” to generate an immune complex. Furthermore,
"Measurement substance" includes antibodies against "measurement substance" (animal species A
Produced by the above) is bound to a water-insoluble carrier, an antibody against the "measurement substance" (produced by animal species B), and a labeled antibody against the antibody produced by animal species B Method of producing body [eg WO 80/02
747, EP-119736, etc., and Journal of Histochem Sightchem, 8
Volume, 1131 pages, published in 1979 (GUESDON, JL, TERNYNCK, T., and
AVRAMEAS, S.: J.Histochem.Cytochem., 8: 1131,1979), etc.]; "Labeling substance" in which an antibody against the "measurement substance" is bound to a water-insoluble carrier, biotin and an antibody against the "measurement substance" And a method of reacting labeled avidin with each other to produce an immune complex are known. Also in the heterogeneous competitive immunoassay, the same assay method as the sandwich immunoassay can be performed by changing the substance to be labeled to a substance having the same immunoreactivity as the "measurement substance".

[0003]

Recently, in immunoassays, the demand for further improvement in sensitivity has been increasing.
According to the above method, it is difficult to increase the amount of labeling in the immune complex, and thus it is difficult to make the measurement with higher sensitivity. Further, in the above method, it is necessary to use an antibody that does not cause cross-reactivity between animal species, and if a substance that interferes with the antibody is present in the serum sample, the measured value may show an abnormality. Although the above method can increase the amount of labeling in the immune complex, non-specific adsorption is large, and as a result, highly sensitive measurement cannot be performed.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have used two types of single-stranded nucleic acids having complementary base sequences, one of which is labeled. It was found that the sensitivity was further increased by the immunoassay method using the method, and the present invention was reached. That is, the present invention comprises the following assay methods and assay reagent kits. : An immune complex formed by reacting a substance to be measured (hereinafter referred to as “measurement substance”) with the following “immobilization substance”, the following “first nucleic acid conjugate” and the following “labeled second nucleic acid” ( An immunoassay method characterized by measuring the amount of label in A) or unreacted "labeled second nucleic acid". Immobilized substance: A substance that has a specific binding property to the "measurement substance" (hereinafter referred to as "binding substance") and a water-insoluble carrier. First nucleic acid conjugate: a conjugate of a substance having the same immunoreactivity as the "binding substance" or "measurement substance" and a single-stranded deoxyribonucleic acid or ribonucleic acid (hereinafter referred to as "first nucleic acid"). . Labeled second nucleic acid: A substance obtained by binding a labeling agent to a single-stranded deoxyribonucleic acid or ribonucleic acid (hereinafter referred to as "second nucleic acid") having a base sequence complementary to the "first nucleic acid". : An immunoassay reagent kit containing the above-mentioned "immobilization substance", "first nucleic acid conjugate" and "labeled second nucleic acid" as components.

In the present invention, the "measurement substance" is
There is no particular limitation as long as an immunoassay can be used. For example, haptens (digoxin, thyroxine, triiodothyronine, cortisol, etc.), hormones (thyroid stimulating hormone, luteinizing hormone, insulin, growth hormone, etc.), serum proteins (immunoglobulin, C-
Reactive proteins), tumor-associated antigens (α-fetoprotein, carcinoembryonic antigen, fetal ferritin, etc.), viruses and virus-related antigens (rubella virus, herpes virus, HBs antigen, HBc antigen, etc.).

Examples of the water-insoluble carrier in the "immobilization substance" include inorganic silicate carriers (glass, silica gel, etc.) and organic carriers (plastic, nitrocellulose, dextran, etc.). Those having a functional group such as an amino group, a thiol group, a carboxyl group, and an active ester group on the surface are preferable because they are easily bound to the "binding substance". In addition, its shape is particle, sheet,
Although it may have any shape such as a tube shape, a more preferable example is a fine particle shape having a particle size of about 0.1 to 50 μm.

The "first nucleic acid" and "second nucleic acid" used in the present invention are both single-stranded nucleic acids selected from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and have complementary base sequences. Have a relationship. Therefore, “first
The "nucleic acid" and the "second nucleic acid" are capable of hybridizing. As the "first nucleic acid" and the "second nucleic acid", any single-stranded DNA or RNA can be used as long as they are complementary sequences, but a base sequence that does not cause a loop structure in the molecule is preferable. . A preferred example is one in which the type of ribonucleotide or deoxyribonucleotide unit constituting the "first nucleic acid" is one. This unit includes adenylic acid, uridylic acid, guanylic acid, cytidylic acid, deoxythymidylic acid, deoxyadenylic acid,
Examples include deoxyguanylic acid and deoxycytidylic acid,
Therefore, one selected from these is preferable.
In addition, the “first
The "second nucleic acid" has an advantage that it can be bound to any part of the "first nucleic acid" as opposed to the "nucleic acid". In addition, "second
The base sequence of the “nucleic acid” is complementarily determined corresponding to the base sequence of the “first nucleic acid”.

The number of ribonucleotide units or deoxyribonucleotide units constituting the "first nucleic acid" and the "second nucleic acid" is preferably in the range of 10 to 1,000. 10
If it is less, the binding force of hybridization is low, 1,
If it exceeds 000, the molecular weight is too large and the reactivity is lowered, which is not preferable.

The "second nucleic acid" may be a single-stranded DNA or RNA corresponding to only one part of the "first nucleic acid", that is, a nucleic acid shorter than the "first nucleic acid" can be used. Therefore, a plurality of "second nucleic acids" can be included in one "first nucleic acid".
There is an advantage that "nucleic acid" can bind. Particularly preferably, the "first nucleic acid" is a nucleic acid composed of one type of nucleotide, and the shorter "second nucleic acid" is
Since it can bind to any part of the “first nucleic acid”, many molecules can efficiently bind to the “first nucleic acid”. That is, since the immunocomplex (A) is produced in a form in which the "labeled second nucleic acid" is bound to the "first nucleic acid conjugate" in a larger amount, the measurement sensitivity is improved. Preferred, "Second
The chain length of the "nucleic acid" is within the range of 1/100 to 1/2 of the "first nucleic acid".

The "first nucleic acid" and "second nucleic acid" can be used by being synthesized or extracted from a living body by a conventionally known method.
Preferred is a nucleic acid prepared by a synthetic method, in which a functional group such as an amino group is introduced at the end thereof.
The introduced functional group is convenient for binding to a substance having the same immunoreactivity as the "binding substance" or "measurement substance", and a labeling agent.

"Immobilization substance" can be performed by a conventionally known method. For example, there are a method of physically adsorbing a protein on a polystyrene carrier, and a method of covalently bonding an amino group and the like of a "binding substance" to a functional group on the surface of a water-insoluble carrier. After binding, the surface of the carrier and unreacted functional groups on the surface are usually blocked with an appropriate protein, nucleic acid, detergent or the like.

The "binding substance" has a specific binding property with respect to the "measurement substance", and includes an antibody, an antigen,
Examples include lectin and protein A, but they can be freely selected within a range that can correspond to the “measurement substance”. Furthermore, the “binding substance” used for the “immobilization substance” and the “first nucleic acid binding substance” does not necessarily have to be the same substance. Preferred examples of the “binding substance” include:
A monoclonal antibody against the "measurement substance" can be mentioned. In this case, it is more preferable that the monoclonal antibodies used for the “immobilization substance” and the “first nucleic acid conjugate” are antibodies having different recognition sites for the “measurement substance”.

The substance having the same immunoreactivity as the "measurement substance" is the same substance as the "measurement substance", a fragment of the "measurement substance" having the immunoreactivity of the "measurement substance", and the "measurement substance". And a substance that does not participate in the reaction between the binding substance and a substance to be measured, and the like.

The "first nucleic acid-conjugated substance", ie, the "binding substance" or the substance having the same immunoreactivity as the "measurement substance" and the "first nucleic acid" is a conventionally known protein molecule. Can be prepared using the crosslinking method of. For example,
By introducing a thiol group into the "binding substance" and binding the amino group introduced into the "first nucleic acid" with a two-crosslinking reagent, a "first nucleic acid conjugate" can be formed.

When the "first nucleic acid conjugate" is a substance obtained by binding the "binding substance" and the "first nucleic acid", the method of the present invention is a method based on the principle of sandwich immunoassay. Further, when the “first nucleic acid conjugate” is a substance obtained by binding the “first nucleic acid” with a substance having the same immunoreactivity as the “measuring substance”, the method of the present invention is based on the competitive immunoassay. Is the way.

Examples of labeling substances (labeling agents) used in the "labeled second nucleic acid" include, for example, isotopes, enzymes,
Examples include fluorescent substances and luminescent substances. Any of these may be known ones. For example, the isotope may be ¹²⁵ I, the enzyme may be horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc., the fluorophore may be a europium derivative, and the luminescent material may be an N-methylacridinium derivative.

The "labeled second nucleic acid" can be prepared by applying a conventionally known binding method for a protein and a labeling agent in any of the above types. For example, as a method of labeling with an enzyme, Nakane et al. Histochem. Sightchem. , 22; 1084, 1974 and the like. As a method of labeling with a fluorescent substance, eye. Hemira, S. Dabuk, Etol. Anal. Biochem. , 137: 335, 1984 and the like. As a method of labeling with a light-emitting body, there are A.I. Wood head, eye. Weeks; Klin. Chem. 29: 1480, 1983 and the like.

The reaction sequence and conditions in the present invention are described below. The reaction may be performed in various orders, but as a preferred example, the “measurement substance” is reacted with the “first nucleic acid binder” and the “immobilization substance” sequentially or simultaneously, and then the “labeled second substance” is added. The immune complex (A) is obtained by reacting “nucleic acid”; “immobilization substance” is reacted with “measurement substance”, and then “first nucleic acid conjugate” and “labeled second nucleic acid” are sequentially added. Alternatively, the immune complex (A) is obtained by reacting simultaneously; the “immobilization substance”, the “first nucleic acid binding substance” and the “labeled second nucleic acid” are simultaneously reacted with the “measuring substance” to obtain the immunity. For example, the complex (A) is obtained; the "measurement substance" is reacted with the "first nucleic acid conjugate" and the "labeled second nucleic acid" sequentially or simultaneously, and then the "immobilization substance" is further reacted.
The order of introducing the constituent components in each reaction, the removal of unreacted substances, and the like can be appropriately performed.

The temperature at which the reaction is carried out, the composition of the solution at which each reaction is carried out, the pH and the like can be carried out under the conditions usually used in immunoassays and hybridization assays. For example, the temperature is preferably 5 to 60 ° C., the pH is preferably 5 to 9, and the solution for carrying out the reaction is preferably an appropriate buffer solution to which salts, proteins, a surfactant and the like are added, if necessary.

As a result of the reaction, the labeling agent is present in the produced immune complex (A) in the amount of the "measurement substance". After removing the unreacted material, by measuring the amount of the labeling agent, or by measuring the amount of the labeling agent in the unreacted material,
It is possible to determine the amount of "measurement substance". The amount of the labeling agent can be measured by various methods depending on the type of the labeling agent, and any method can be freely selected by those skilled in the art. By comparing the measured labeled amount with the labeled amount when a known amount of the “measured substance” is measured, the amount of the measured substance in the sample can be determined.

[0021]

The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

Example 1 This example illustrates the use of α-fetoprotein (A
FP) was measured using synthetic DNAs having different lengths.

1) Synthesis of polydeoxyadenylic acid and polydeoxythymidylate Using an automatic DNA synthesizer (Model 392, manufactured by Applied Biosystems Japan Ltd.), nucleotides were prepared according to the instructions attached to the device. Polydeoxyadenylic acids of the formulas 10, 50 and 100 were respectively synthesized, and an amino group was further introduced to the 5 ′ end by an aminohexyl linker (Aminolink 2 manufactured by Applied Biosystems Japan Ltd.). The synthesized amino group-introduced polydeoxyadenylic acid having each chain length was purified by a column, dissolved in distilled water, and used in the next operation. Similarly, the number of nucleotides is 10,
50,100 amino group-introduced polydeoxythymidylate were synthesized.

2) Preparation of Polydeoxyadenylic Acid-Binding Antibody From anti-AFP antibody (manufactured by Dako), E. Ishikawa, M. Imagawa, etor. , J. Immunoassay,
4; 209, 1983, and Fab ′ antibody was prepared. Amino group-introduced polydeoxyadenylic acid solution (nucleic acid amount: 1 mg) of each chain length synthesized in 1) and Fab ′ antibody (10 mg) were combined with a phosphate buffer solution (0.1 mol, pH: 6.
0, 1 mM EDTA) in 2 ml, mix and thaw,
0.2 mg of 2 cross-linking reagent N- (γ-maleimidobutyryloxy) succinimide (GMBS; manufactured by Dojindo Chemical Co., Ltd.) dissolved in 0 μl of dimethylformamide was added at 30 ° C.
And reacted for 2 hours. Unreacted GM dialyzed against phosphate buffer
BS was removed to obtain a polydeoxyadenylic acid-conjugated anti-AFP antibody. It was stored at 4 ° C. and diluted with a phosphate buffer containing 0.1% bovine serum albumin before use.

3) Preparation of anti-AFP antibody-bonded fine particles Copolymer particles having an active ester group on the surface (ASU copolymer manufactured by Kokusan Kagaku Co., Ltd .; average particle diameter 1 μm) 1
0 mg to 2 ml of phosphate buffer (0.02 mol, pH
It was washed twice with 7.2) and centrifuged, and suspended in 0.5 ml of a phosphate buffer. 1 mg of anti-AFP antibody (manufactured by Dako) was added to the fine particle suspension, and the mixture was reacted at room temperature for 8 hours. After washing twice with a phosphate buffer containing 0.1% bovine serum albumin,
The cells were suspended in 2 ml of a phosphate buffer containing 1% bovine serum albumin and stored at 4 ° C until use.

4) Preparation of polydeoxythymidylate-bound peroxidase 1 mg of horseradish peroxidase (manufactured by Toyobo) was added to phosphate buffer (0.1 mol, pH 6.0, 1 mM EDT).
It melted in 1 ml (containing A), 0.1 mg of 2-iminothiolane hydrochloride was added, and the mixture was reacted at 37 ° C. for 30 minutes. The reaction solution was applied to a gel filtration column to remove unreacted 2-iminothiolane, to obtain a thiol group-introduced peroxidase.

2 A cross-linking reagent N- dissolved in dimethylformamide was added to a polydeoxythymidylic acid solution containing an amino group.
(Γ-maleimidobutyryloxy) succinimide (G
MBS; manufactured by Dojindo Co., Ltd.) (0.2 mg) and added at 30 ° C. for 2
Reacted for hours. Unreacted GMBS dialyzed against phosphate buffer
Was removed. A thiol group-introduced peroxidase solution was added and reacted at 4 ° C. for 8 hours to obtain polythymidylate-bound peroxidase. It was stored at 4 ° C. and diluted with a phosphate buffer containing 0.1% bovine serum albumin before use.

5) Measurement procedure 50 μl of serum sample and 50 μl of polydeoxyadenylic acid-conjugated anti-AFP antibody and 5 μl of anti-AFP antibody-conjugated fine particle suspension were reacted in a test tube at 37 ° C. for 15 minutes, and then distilled water (2 ml) was added. The microparticles were washed 3 times and centrifuged. Discard the supernatant,
100 μl of a diluted polydeoxythymidylate-bound peroxidase solution was added, and the particles were resuspended and reacted at 37 ° C. for 15 minutes, and then the microparticles were washed 3 times with 2 ml of distilled water and centrifuged. The supernatant is discarded, 0.5 ml of a substrate solution (hydrogen peroxide-containing O-phenylenediamine solution) is added, and the particles are resuspended.
The reaction was carried out at 7 ° C for 15 minutes. Centrifuge and collect the absorbance of the supernatant 492n
It was measured in m. The AFP concentration in the sample was read from the calibration curve prepared by similarly measuring the standard solution containing the known concentration of AFP. As the standard solution, a commercially available AFP measurement kit “Grazyme AFP-EIA TEST” (sold by Wako Pure Chemical Industries, Ltd.) was used.

6) Measurement Results Table 1 shows the results of measuring standard AFP by combining each of the polydeoxyadenylate-conjugated anti-AFP antibody and the polydeoxythymidylate-conjugated peroxidase having different nucleotide numbers. When the chain length of the nucleic acid bound to the antibody is short,
Although the sensitivity is slightly low, an improvement in the sensitivity is recognized when the nucleic acid bound with the label is shorter than the chain length of the nucleic acid bound to the antibody.

[0030]

[Table 1]

Table 2 shows the results of measurement of serum specimens of normal subjects and liver cancer patients with a combination of 100 and 10 nucleotides.

[0032]

[Table 2]

COMPARATIVE EXAMPLE 1 This comparative example shows the measurement by the conventional method.

1) Preparation of avidin-bound peroxidase 1 mg of horseradish peroxidase (manufactured by Toyobo Co., Ltd.) was added to phosphate buffer (0.1 mol, pH 6.0, 1 mM EDT).
It melted in 1 ml (containing A), 0.1 mg of 2-iminothiolane hydrochloride was added, and the mixture was reacted at 37 ° C. for 30 minutes. The reaction solution was applied to a gel filtration column to remove unreacted 2-iminothiolane, to obtain a thiol group-introduced peroxidase. Phosphate buffer containing 1 mg of egg white avidin (manufactured by Sigma) (0.
1M, pH 6.0) 2 cross-linking reagent N- (γ-maleimidobutyryloxy) succinimide (GMBS; Dojindo) 0.2 dissolved in dimethylformamide 0.2
mg was added and reacted at 30 ° C. for 2 hours. The unreacted GMBS was removed by dialysis with a phosphate buffer. After adding a thiol group-introduced peroxidase solution and reacting at 4 ° C. for 8 hours, an unreacted material was removed through a gel filtration column to obtain an avidin-bonded peroxidase. Store at 4 ℃ and use
It was diluted with a phosphate buffer containing 0.1% bovine serum albumin before use.

2) Preparation of biotinylated anti-AFP antibody Phosphate buffer 1 containing 1 mg of anti-AFP antibody (manufactured by Dako)
To ml, NHS-biotin (N-Hydroxysu
Ccinimido-biotin; manufactured by PIERCE) dimethylformamide 20 μ dissolved in 0.5 mg
1 was added and reacted at 37 ° C. for 90 minutes. Unreacted substances were removed by gel filtration, and the antibody fraction was collected and used as a biotinylated antibody. It was diluted with a phosphate buffer containing 1% BSA and used.

3) Measurement operation Biotinylated anti-AFP antibody 5 diluted with 50 μl of serum sample
After reacting 0 μl and 5 μl of avidin-bonded fine particle suspension in a test tube at 37 ° C. for 15 minutes, the fine particles were washed 3 times with 2 ml of distilled water and centrifuged. The supernatant was discarded, 100 μl of diluted peroxidase-labeled anti-AFP antibody solution was added, the particles were resuspended and reacted at 37 ° C. for 15 minutes, and then the microparticles were washed 3 times with 2 ml of distilled water and centrifuged. The supernatant was discarded, 0.5 ml of a substrate solution (hydrogen peroxide-containing O-phenylenediamine solution) was added, and the particles were resuspended and reacted at 37 ° C. for 15 minutes. After centrifugation, the absorbance of the supernatant was measured at 492 nm. The AFP concentration in the sample was read from the calibration curve prepared by similarly measuring the standard solution containing the known concentration of AFP.

4) Measurement Results The same measurement results as in Example 1 for standard solutions, serum samples from normal subjects and liver cancer patients are shown in Table 2 above. Since the background (absorbance at standard 0 concentration) is higher than that in Example 1 and the measured absorbance is low, the measurement sensitivity is poor. Therefore, the measured value of normal human serum is inaccurate. Moreover, there were clinically problematic results because some specimens showed abnormally high values even though they were normal human sera, and some showed low values even in patient sera.

Example 2 This example is an example of ferritin measurement according to the present invention using a gene isolated from a living body.

1) Purification of phage DNA Using the M13 cloning kit (manufactured by Toyobo Co., Ltd.) and following the instructions attached to the kit, the phage double chain D was isolated from the bacterial cells.
The phage 1 heavy chain DNA was purified from NA and culture supernatant.
M13 phage has about 7,000 nucleotides (7 Kb)
It is composed of

2) Preparation of anti-ferritin antibody bound to 1-heavy chain DNA The 1-heavy chain DNA purified in 1) was mechanically stirred and then subjected to sucrose density gradient centrifugation to obtain a 1 Kb-long fraction. . To 10 μg of this single-stranded DNA was dissolved in 1 ml of phosphate buffer (0.1 mol, pH 6.0), 5 μg of 2-iminothiolane hydrochloride was added and reacted at 37 ° C. for 30 minutes to introduce a thiol group. did. A 3-fold amount of ethanol was added to precipitate and collect the DNA, which was again dissolved in 1 mM EDTA-containing phosphate buffer to obtain a thiol group-introduced single-stranded DNA.

To 1 ml of a phosphate buffer (0.1 mol, pH 6.0) containing 1 mg of anti-ferritin antibody (manufactured by Dako),
0.5 mg of GMBS dissolved in dimethylformamide was added and reacted at 30 ° C. for 90 minutes. After dialyzing against phosphate buffer to remove unreacted GMBS, mixed with thiol group-introduced single-stranded DNA solution and reacted for 8 hours, then unreacted by gel filtration column to remove single-stranded It was a DNA-binding anti-ferritin antibody.

3) Preparation of ³² P-labeled DNA The double-stranded DNA (RF-DNA) purified in 1) was added to Maniatis, Etol. , Molecular Cloning: 109-112, using a nick-ray Deployment Method according to Cold Spring Harbor Laboratory Lee (1982), was introduced ³² P. After introduction, RF-DNA
Was disrupted by ultrasonic waves, and two fractions having chain lengths of 500 to 300 bp and 200 to 50 bp or less were obtained by agarose gel electrophoresis. At the time of use, it was heated at 70 ° C. and used as ³² P-labeled single-stranded DNA.

4) Preparation of anti-ferritin antibody-bonded fine particles Copolymer particles having an active ester group on the surface (Kokusan Kagaku Co., Ltd., ASU copolymer; average particle diameter 1 μm) 1
0 mg to 2 ml of phosphate buffer (0.02 mol, pH
It was washed twice with 7.2) and centrifuged, and suspended in 0.5 ml of a phosphate buffer. 1 mg of anti-ferritin antibody (manufactured by Dako) was added to the fine particle suspension, and the mixture was reacted at room temperature for 8 hours. After washing twice with a phosphate buffer containing 0.1% bovine serum albumin,
It was suspended in 2 ml of a phosphate buffer containing 0.1% bovine serum albumin and stored at 4 ° C until use.

5) Measurement procedure 50 μl of standard ferritin solution and 50 μl of diluted single-stranded DNA-binding anti-ferritin antibody and 5 μl of anti-ferritin antibody-bound fine particle suspension were reacted in a test tube at 37 ° C. for 15 minutes, and then distilled water (2 ml). The microparticles were washed 3 times and centrifuged. ³² P-labeled DNA solution 100, which was prepared by diluting and heating the supernatant
After adding μl, the particles were resuspended and reacted at 37 ° C. for 30 minutes, and then the particles were washed 3 times with 2 ml of distilled water and centrifuged. The supernatant was discarded and the count from ³² P was measured with a scintillation counter. As the standard ferritin, a commercially available ferritin measurement kit "Glaozyme Ferritin" (sold by Wako Pure Chemical Industries, Ltd.) was used.

6) Measurement Results The calibration curves for ferritin in this example are shown in FIGS. 1 and 2. FIG. 1 shows that the ³² P-labeled DNA has a chain length of 500 to 300 b.
The results are for p. FIG. 2 shows the results when the chain length of ³² P-labeled DNA is 200 to 50 bp. The shorter the chain length, the higher the measurement sensitivity.

Example 3 This example is an example of a competitive immunoassay for thyroxine (T4) according to the present invention.

1) Preparation of polydeoxyadenylic acid-bound thyroxine 1 ml of dimethylformamide was added to 1 mg of T4 (manufactured by Calbio Chemistry) and dissolved. 1 ml of this solution
After adding 0.2 mg of 2-iminothiolane hydrochloride to the mixture and reacting at room temperature for 1 hour, unreacted 2-iminothiolane hydrochloride was removed by gel filtration to obtain thiol group-introduced T4. GMBS 0.1 m prepared by dissolving the amino group-introduced polydeoxyadenylic acid (100 nucleotides) prepared in Example 1 in a phosphate buffer and dissolving it in dimethylformamide.
After the reaction was carried out at 37 ° C. for 90 minutes, unreacted GMBS was removed by gel filtration to obtain a nucleic acid fraction. Thiol group-introduced T4 was added to the nucleic acid fraction, and after reacting at 37 ° C. for 90 minutes, unreacted substances were removed by gel filtration to obtain polydeoxyadenylic acid-bound thyroxine.

2) Preparation of anti-T4 antibody-bonded fine particles Copolymer particles having an active ester group on the surface (ASU copolymer manufactured by Kokusan Kagaku Co., Ltd .; average particle diameter 1 μm) 1
0 mg to 2 ml of phosphate buffer (0.02 mol, pH
It was washed twice with 7.2) and centrifuged, and suspended in 0.5 ml of a phosphate buffer. 1 mg of anti-T4 antibody (manufactured by Dako) was added to the fine particle suspension, and the mixture was reacted at room temperature for 8 hours. After washing twice with a phosphate buffer containing 0.1% bovine serum albumin, 0.1
Suspended in 2 ml of phosphate buffer containing% bovine serum albumin,
Stored at 4 ° C until use.

3) Measuring method Standard T4 solution (T4 dissolved in T4 free serum)
50 μl, 5 μl of anti-T4 antibody-bound microparticle suspension and 50 μl of diluted polyadenylate-bound thyroxine solution were reacted at 37 ° C. for 15 minutes. After the reaction, the fine particles were washed twice with 2 ml of distilled water and centrifuged. The supernatant is discarded, and 200 μl of a diluted solution of polydeoxythymidylate (number of nucleotides 50) -bound peroxidase prepared in Example 1 is added.
The reaction was performed at 15 ° C for 15 minutes. After the reaction, the fine particles were washed twice with 2 ml of distilled water and centrifuged. The supernatant was discarded, 0.5 ml of a substrate solution (hydrogen peroxide-containing O-phenylenediamine solution) was added, and the particles were resuspended and reacted at 37 ° C. for 15 minutes. After centrifugation, the absorbance of the supernatant was measured at 492 nm.

4) Measurement Results The calibration curve for T4 measurement in this example is shown in FIG. Sufficient measurement sensitivity was shown under the measurement conditions for a short time.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, 2 having a complementary nucleotide sequence
This is an immunoassay method that utilizes the hybridization of a single-stranded nucleic acid of a book. Conventionally, in the immunoassay, various methods have been performed in order to perform highly sensitive measurement, but sufficient effects have not been obtained. That is, there has been no method that simultaneously achieves a sufficient increase in the amount of labeling in the immune complex and a reduction in the background. The present invention can increase the amount of labeling in the immune complex by utilizing the hybridization of nucleic acids. In particular, by reacting a single-stranded nucleic acid with a shorter single-stranded nucleic acid, it has become possible to significantly increase the amount of labeled label and improve the measurement sensitivity. In addition, the low measurement background also improves the accuracy of low values. Further, it is less susceptible to the interfering substances present in the sample, and does not show clinically erroneous measurement values. As described above, the present invention provides a method and a measuring reagent that enable highly sensitive and accurate immunoassay.

[Brief description of drawings]

FIG. 1 shows that in Example 2, the chain length is 500 to 300 bp.
It is a calibration curve when standard ferritin is measured using ³² P-labeled DNA.

[Fig. 2] In Example 2, ³² having a chain length of 200 to 50 bp
It is a calibration curve when standard ferritin is measured using P-labeled DNA.

FIG. 3 is a calibration curve when a standard T4 solution was measured in Example 3.

Claims (6)

[Claims] 1. A substance to be measured (hereinafter referred to as “measurement substance”) is produced by reacting the following “immobilization substance”, the following “first nucleic acid conjugate” and the following “labeled second nucleic acid”. An immunoassay method characterized by measuring the amount of label in the immune complex (A) or in the unreacted "labeled second nucleic acid". Immobilized substance: A substance that has a specific binding property to the "measurement substance" (hereinafter referred to as "binding substance") and a water-insoluble carrier. First nucleic acid conjugate: a conjugate of a substance having the same immunoreactivity as the "binding substance" or "measurement substance" and a single-stranded deoxyribonucleic acid or ribonucleic acid (hereinafter referred to as "first nucleic acid"). . Labeled second nucleic acid: A substance obtained by binding a labeling agent to a single-stranded deoxyribonucleic acid or ribonucleic acid (hereinafter referred to as "second nucleic acid") having a base sequence complementary to the "first nucleic acid". 2. The method according to claim 1, wherein the type of unit selected from ribonucleotides or deoxyribonucleotides constituting the "first nucleic acid" is one type. 3. The method according to claim 1, wherein the “binding substance” is an antibody or an antigen against the “measurement substance”. 4. The number of units selected from ribonucleotides or deoxyribonucleotides constituting the “first nucleic acid” and the “second nucleic acid” is 10 to 1,000, respectively. Method. 5. The number of units selected from ribonucleotides or deoxyribonucleotides constituting the “second nucleic acid” is 1 times the number of similar units constituting the “first nucleic acid”.
/ 100 to 1/2. The method according to any one of claims 1 to 4. 6. The “immobilization substance”, the “first nucleic acid conjugate” and the “labeled second nucleic acid” according to any one of claims 1 to 5.
An immunoassay reagent kit containing as a component.